β-Lactam antibiotics constitute the most important group of antibiotic compounds, with a long history of clinical use. Among this group, the prominent ones are the penicillins and cephalosporins. These compounds are naturally produced by the filamentous fungi such as Penicillium chrysogenum and Acremonium chrysogenum, respectively.
As a result of classical strain improvement techniques, the production levels of the antibiotics in P. chrysogenum and A. chrysogenum have increased dramatically over the past decades. With the increasing knowledge of the biosynthetic pathways leading to penicillins and cephalosporins, and the advent of recombinant DNA technology, new tools for the improvement of production strains and for the in vivo derivatisation of the compounds have become available.
Most enzymes involved in β-lactam biosynthesis have been identified and their corresponding genes been cloned, as can be found in Ingolia and Queener, Med. Res. Rev. 9 (1989), 245–264 (biosynthesis route and enzymes), and Aharonowitz, Cohen, and Martin, Ann. Rev. Microbiol. 46 (1992), 461–495 (gene cloning).
The first two steps in the biosynthesis of penicillin in P. chrysogenum are the condensation of the three amino acids L-5-amino-5-carboxypentanoic acid (L-α-aminoadipic acid) (A), L-cysteine (C) and L-valine (V) into the tripeptide LLD-ACV, followed by cyclisation of this tripeptide to form isopenicillin N. This compound contains the typical β-lactam structure.
The third step involves the exchange of the hydrophilic side chain of L-5-amino-5-carboxypentanoic acid by a hydrophobic side chain by the action of the enzyme acyltransferase. The enzymatic exchange reaction mediated by acyltransferase takes place inside a cellular organelle, the microbody, as has been described in EP-A-0 448 180.
Cephalosporins are much more expensive than penicillins. One reason is that some cephalosporins (e.g. cephalexin) are made from penicillins by a number of chemical conversions. Another reason is that, so far, only cephalosporins with a D-5-amino-5-carboxypentanoyl side chain could be subjected to fermentative conversion. Cephalosporin C, by far the most important starting material in this respect, is very soluble in water at any pH, thus implying lengthy and costly isolation processes using cumbersome and expensive column technology. Cephalosporin C obtained in this way has to be converted into therapeutically used cephalosporins by a number of chemical and enzymatic conversions.
The methods current favoured in industry to prepare the intermediate 7-ADCA involve complex chemical steps leading to the expansion and derivatisation of penicillin G. One of the necessary chemical steps to produce 7-ADCA involves the expansion of the 5-membered penicillin ring structure to a 6-membered cephalosporin ring structure (see for instance U.S. Pat. No. 4,003,894). This complex chemical processing is both expensive and noxious to the environment.
Consequently, there is a great desire to replace such chemical processes with enzymatic reactions such as enzymatic catalysis, preferably in an in vivo process. A key to the replacement of the chemical expansion process by enzymatic and in vivo processes is the central enzyme in the cephalosporin biosynthetic pathway, desacetoxycephalosporin C synthetase (DAOCS), also called “expandase”.
The expandase enzyme from the bacterium Streptomyces clavuligerus was found to carry out, in some cases, penicillin ring expansions. When introduced into P. chrysogenum, it can convert the penicillin ring structure into the cephalosporin ring structure in vivo, as described in Cantwell et al., Proc. R. Soc. Lond. B. 248 (1992), 283–289. The expandase enzyme has been well characterised (EP-A-0 366 354) both biochemically and functionally, as has its corresponding gene, cefE. Both physical maps of the cefE gene (EP-A-0 341 892). DNA sequence and transformation studies in P. chrysogenum with cefE have been described.
Another source for a ring expansion enzyme is for example the bacterium Nocardia lactamdurans (formerly Streptomyces lactamdurans) and the fungus Acremonium chrysogenum (formerly Cephalosporium acremonium). The expandase from Cephalosporium is a bifunctional enzyme, having expandase (ring-expansion) as well as 3-hydroxylating activity. Both the biochemical properties of the enzyme and the DNA sequence of the gene have been described (Cortés et al., J. Gen. Microbiol. 133 (1987), 3165–3174; and Coque et al., Mol. Gen. Genet. 236 (1993), 453–458, respectively).
Since the expandase catalyses the expansion of the 5-membered thiazolidine ring of penicillin N to the 6-membered dihydrothiazine ring of DAOC this enzyme would be a logical candidate to replace the ring expansion steps of the chemical process. Unfortunately, the enzyme works on the penicillin N intermediate of the cephalosporin biosynthetic pathway, but not, or relatively inefficiently, on the readily available inexpensive penicillins as produced by P. chrysogenum, like penicillin V or penicillin G. Penicillin N is commercially not available and even when expanded, its D-5-amino-5-carboxypentanoyl side chain cannot be easily removed by penicillin acylases.
It has recently been found that the expandase enzyme is capable of expanding penicillins with non-naturally occurring side chains to the corresponding 7-ADCA derivatives. These side chains include adipate (5-carboxyl-pentanoic acid) and various other compounds. This feature of the expandase has been exploited in a process for the in vivo production of adipoyl-7-ADCA in Penicillium chrysogenum as disclosed in EP-A-0 532 341. Penicillium chrysogenum strains are transformed with the Streptomyces clavuligerus expandase to produce adipoyl-6-aminopenicillanic acid (adipoyl-6-APA) when these transformants are fed adipic acid. Subsequently, adipoyl-6-APA is “expanded” to adipoyl-7-ADCA. Production of various other 7-acylated cephalosporins have been disclosed in WO 95/04148 and WO 95/04149. In WO 95/04148 and WO 95/04149 it has been disclosed that addition of 3′-carboxymethyl thiopropionic acid and 3,3′-thiodipropionic acid to the medium yield penicillins that are substrates for the expandase, leading to the synthesis of 2-(carboxyethylthio)acetyl-7-ADCA and 3-(carboxymethylthio)propionyl-7-ADCA, respectively.
Even though expandase displays an activity on penicillins with different side-chains, the expansion of these novel penicillins occurs less efficient as compared to the expansion of the natural substrate, penicillin N. The notion has been the basis for modifying the expandase, aiming to alter the activity on adipoyl-penicillanic acid. Application of mutagenised expandase has been disclosed in WO 98/02551.
The final step in the synthesis of penicillins, the exchange of the α-amino adipoyl side chain of IPN by an alternative side-chain, occurs in the microbody, where the acyltransferase is localised. For optimal expansion of the penicillin, the preferred localisation of expandase is therefore believed to be in the microbody. This is based not only on the fact that the substrate for expandase is believed to be produced in the microbody, but also on the expectation that in the microbody, expandase should be better protected from degradation by proteases, which is a known drawback from the use of P. chrysogenum for the production of enzymes (see for example, Theilgaard et al., 1997; Purification and characterisation of δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase from Penicillium chrysogenum; Biochem. J. 327, 185–191).
Modification of cellular localisation of enzymes involved in beta-lactam antibiotic production has been suggested in EP 0 448 180. For example on page 7, penultimate paragraph, it is suggested to localise epimerase and preferably subsequent cephalosporin biosynthetic enzymes like expandase/hydroxylase from Acremonium chrysogenum or expandase from Streptomyces spec. in the microbodies, to improve the efficiency of the production of cephalosporin or intermediates in Penicillium chrysogenum. It was also shown therein, that removal of the microbody targeting signal of acyltransferase, an enzyme involved in the removal of the side chain of acyl-6-APA destroys penicillin production altogether. This strongly suggests, that enzymes involved in beta-lactam production should be localised in the microbodies, if not for reasons of substrate localisation then at least for reasons of stability.